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DNA Detectives

By Suzanne Black



This lab was created in 1990 by Lane Conn, San Francisco State. I worked with Jim Carmack (Lowell HS, San Francisco) to flesh it out and field-test it.


Type of Activity:

  • Hands-on laboratory, both simulation( we use lambda phage DNA, not human DNA and inquiry-based (whose DNA was recovered from the crime scene?)
  • The question this lab helps students answer is: How is DNA manipulated and analyzed as forensic evidence? More to the point: 'Who Dunnit?'

Target Audience:

  • Biology
  • Genetics
  • Integrated social studies/biology block

Required of the students:

Prior practice with micropipettors; familiarity with the roles and responsibilities of group work.

Preparation time:

This lab requires attention to detail, but it's worth it. Aliquot all your reagents ahead of time into labeled sets of tubes so YOU won't get mixed up! Place supplies around the classroom to avoid congestion and long lines.

Class time needed:

One week (five 50-min. lab periods) MINIMUM; longer (2-3 weeks) if you include crime scene development, micropipeting practice, or extension activities.


Background

  • The students think they have different DNA and the same enzyme, but you:
    • Give Group 1 lambda DNA ("suspect 1 DNA") and ECoRI enzyme,
    • Give Group 2 lambda DNA ("suspect 2 DNA") and HindIII enzyme,
    • Give Group 3 lambda DNA ("suspect 3 DNA") and BAmHI enzyme,
    • Give Group CS lambda DNA ("crime scene DNA:) and HindIII enzyme.

    I also assign a fifth group, Group R, to prepare a reference sample using lambda DNA ("crime scene DNA") and water, instead of enzyme.

  • The five large groups described above (6-7 students each) need exist only on Day 1.

    They make multiple tubes containing the same reaction mixture. For the remainder of the lab, students can work in 2's or 3's.

  • Devise a crime scenario to introduce the lab and the three suspects. This year, we investigated a fictitious jewelry store robbery in a local mall. Invent a motive, opportunity, and alibi for each suspect. Require students to hypothesis who committed the crime before they analyze the DNA.


Project

DNA Detectives

Introduction: Many of the revolutionary changes that have occurred in biology over the past fifteen years can be attributed directly to the ability to manipulate DNA in defined ways. The principal tools for this recombinant DNA technology are enzymes that can "cut and "paste" DNA. Restriction enzymes are the "chemical scissors" of the molecular biologist; these enzymes cut DNA at specific nucleotide sequences. A sample of someone's DNA, incubated with restriction enzymes, is reduced to millions of DNA fragments of varying sizes. A DNA sample from a different person would have a different nucleotide sequence and would thus be enzymatically "chopped up" into a very different collection of fragments. We have been asked to apply DNA fingerprinting to determine which suspect should be charged with a crime perpetrated in our city.

Purpose:

To prepare and analyze a DNA fingerprint, the student will:

  • cut a DNA sample -- the evidence! -- by incubating it with restriction enzymes,

  • load an agarose gel with the restriction digest,

  • conduct gel electrophoresis to sort out the DNA fragments in the digest,

  • staining and photograph the gel to visualize the DNA, and

  • analyze the resulting banding pattern or "DNA fingerprint" to solve a crime.

Materials, per team:

  • electrophoresis box
  • casting tray
  • comb
  • agarose solution [0.8% in TAE]
  • at 65degrees-C
  • power supply
  • restrictions enzymes
  • on ice
  • plastic tray for storing gel
  • DNA from suspect or crime scene
  • assigned
  • P-20 micropipet & tips
  • electrophoresis buffer [1X TAE]
  • racks for 1.5 mL reaction tubes
  • restriction buffer [2X]
  • 1.5mL reaction tubes
  • several
  • loading dye

Class use:

water bath, crushed ice containers, microcentrifuge, documentation station (camera, film, filter, hood, UV transilluminator), DNA staining solution in trays.

Receive DNA Sample ("crime scene" or "suspect"), (DAY 1):

1. You will be assigned to one of five forensic groups. Do not share reagents or equipment with another forensic group.

2. Obtain DNA and reagents as follows:

*Group 1: stock tube: DNA from suspect "1"*Group 2: stock tube: DNA from suspect "2"
stock tube: restriction bufferstock tube: restriction buffer
enzyme (in cooler; ask teacher)enzyme (in cooler; ask teacher)
supply of 1.5 mL reaction tubes supply of 1.5 mL reaction tubes
*Group 3: stock tube: DNA from suspect "3"*CS Group: stock tube: "crime scene" DNA
stock tube: restriction buffer stock tube: "crime scene" DNA
enzyme (in cooler; ask teacher) enzyme (in cooler; ask teacher)
supply of 1.5 mL reaction tubes supply of 1.5 mL reaction tubes
*Group R: stock tube: "crime scene" DNA
stock tube: restriction buffer
stock tube: distilled water
supply of 1.5 mL reaction tubes

Incubate DNA and restriction enzymes at 37-degreesC.

1. Each forensic group will make the restrictions digests for all teams in the class. For example, if there are 10 total teams (30 students) in the lab, Group1 would prepare a total of 10 tubes in which Suspect 1's DNA is mixed with restriction enzyme and buffer.

2. Start by labeling the required number of empty tubes with your forensic group label.
Use a permanent ink marker. Then, in each tube, put:

  • 4 microL of DNA (suspect or crime scene)
  • 5 microL of restriction buffer
  • 2 microL of enzyme. (Reference group substitutes distilled water)

CAUTIONS

WATCH as the tiny volumes go in or out of the pipet.
THINK about what you are doing!
Change tips when you change reagents. Don't contaminate stocks!
DNA and buffer go in a tube before enzyme; add enzyme last.

3. After all three reagents have been added to the tubes, close their caps tightly and mix them by giving them a 2-3 sec. spin in a BALANCED microcentrifuge.

4. Place the tubes in a 37degreeC water bath for 30 min.; then refrigerate until the next day.

Cast an agarose gel (DAY 2): (From now on, work in teams of 2-3 students)

1. Prepare the casting tray ("gates" up, or add scotch tape) and insert a 6-well comb.

2. Obtain a beaker with 20 mL of 65degreeC liquid agarose (CAUTION: HOT!). When the beaker is just cool enough to hold, pour the agarose evenly into the floor of the tray.

3. DO NOT jar or move the casting tray as the gel solidifies. This ensures a smooth, even gel. AS the agarose polymerizes (about 10 min), it changes from clear to slightly opaque.

4. While you are waiting, fill the plastic electrophoresis box with about 300 mL of TAE electrophoresis buffer. (TAE is a buffered salt solution.)

5. When the gel has solidified, lower the "gates" (or, remove tape) on the casting tray and submerge the tray in the gel box. The comb-end should be located at the cathode (negative) electrode. Only a few millimeters of buffer should cover the surface of the gel.

6. Carefully remove the comb from the gel (pull it straight out). You'll notice that this creates six little empty "slots" or wells in the gel.

Load the gel.

1. Each team needs to pick up one of each tube -- that is , a #1, a #2, a #3, a CS, & an R.

2. To each of your five tubes, add 2 microL loading dye. Give them a quick spin in the microfuge to mix the contents.

3. Load 10 microL of each sample into a separate well, CHANGING TIPS BETWEEN SAMPLES!

*Lower the pipet tip under the surface of the buffer, but don't puncture the floor of the gel.
*Gently depress pipet plunger and slowly expel a sample into a well. Keep plunger depressed until the pipet is out of the gel box.

Gel electrophoresis (begin DAY 2, finish DAY 3):

The term 'electrophoresis' literally means "to carry with electricity." It is a technique for separating and analyzing mixtures of charged molecules. When placed in an electric field, pieces of DNA (because they are ionized and negatively charged at physiologic pH) migrate toward the positive electrode (anode); small pieces of DNA experience less resistance and move faster (farther) than large pieces.

CAUTIONS

Remember, it is good practice to turn the power supply OFF before touching or opening gel box.
If two teams are connecting their gel boxes to one power supply, be sure to communicate when turning the power supply ON or OFF.

1. WITH THE POWER SUPPLY OFF, secure the lid of the gel box and connect the leads to the same channel of the power supply (red-red, black-black).

2. Set the power supply at about 100 Volts and 40 milliAmps current. (Eighty milliAmps will automatically result if a second box is connected to one power supply.)

3. Turn the power supply ON. Notice there is a switch to direct the LED display to read in either volts or milliamps. Use it to verify that current is flowing through the gel.

4. Allow electrophoresis to proceed until the dye, and DNA, are out of the wells and securely held by the agarose molecules.

5. CONVENIENT STOP POINT. Turn the power supply OFF. Disconnect the leads.

6. Remove the casting tray from the gel box. Carefully slide your gel into an empty, labeled plastic container. Your teacher will store the gels in the refrigerator overnight.

DAY 3:

1. TO RESUME ELECTROPHORESIS, CAREFULLY return your gel to a casting tray (gates down; no tape).

2. Submerge the tray into a gel box containing buffer; connect the gel box to a power supply; turn the power supply ON; and adjust the voltage as before.

3. Continue to electrophorese until the fastest-moving dye front has advanced at least 2/3 to 3/4 of the way across the gel (about 45 min. total time).

4. Turn the power supply OFF and disconnect the leads.

5. Remove the casting tray from the gel box. CAREFULLY slide your gel off the casting tray and into its labeled plastic container.

Stain and photograph the gel. (DAYS 4 & 5):

There are many stains and tagged probes that allow us to visualize DNA. In our lab, we will stain the DNA with a fluorescent dye called ethidium bromide (EBr). When excited by ultraviolet (UV) light, the EBr absorbs some of the energy and emits orange (visible) light.

CAUTIONS

Do not allow your skin, eyes, mouth, etc. to come in contact with ethidium bromide solution. Always wear goggles and gloves if working with this chemical. UV light can damage unprotected eyes and skin. Never look directly into an unshielded UV light source. Our transilluminators are safe, since they will not turn on unless the plastic safety shield is lowered over the gel.

1. Bring your container to the Staining Station.

2. An operator, wearing gloves, will transfer your gel to a staining dish. The staining dishes contain a dilute EBr solution.

3. The gel will be left in the EBr stain for 5-10 min., rinsed in distilled water for 5 min. (to increase contrast and make the gels safer to handle). and returned to its container.

4. Take your stained, rinsed gel to the Documentation Station.

5. An operator will put your gel on the surface of a UV transilluminator. When the safety-lid is closed, this instrument emits ultraviolet light. The ultraviolet light causes ethidium bromide-coated DNA fragments to glow.

6. The operator takes a Polaroid 667 photograph of your gel for each team member. After about 45 sec. of developing time, peel the backing away to separate the print from the negative. (Caution: Developing fluid is caustic -- don't let it touch you.)

7. EXAMINE YOUR PHOTOGRAPH!

Upon completion of this lab:

  • Dispose of designated materials in the appropriate places.
  • Leave equipment as you found it.
  • Check that your work station is in order.
  • Wash your hands.

Evaluation Ideas: The teacher -- YOU -- can:

1. Lead a class comparison of gel photographs. Photocopy the photographs and make overhead transparencies of the photocopies. Was the DNA cut? Is there a match between any of the suspects' DNA and the crime scene DNA? Are there any unusual DNA patterns, and if so, how might they have resulted?

2. Assign to each team a group lab report. Distribute your grading rubric ahead of time. Designate some class time for peer revision and rewriting.

3. Extend the lab: stage a mock trial which makes use of the students' DNA evidence.

4. Take a field trip to the county courthouse to observe a real trial with real DNA evidence.

Invariably, your students will want to know whose (?) DNA they used. This is a very teachable moment! Now's your chance to compare virus and eukaryote genomes, or to explain why DNA probes, Southern blotting, and autorads are essential for real DNA fingerprinting.


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